Methods for detecting risk of fatal prostate cancer using serum calcium

ABSTRACT

A method of screening for increased risk of fatal prostate cancer in a subject comprises providing a blood sample collected from the subject, and then detecting the presence or absence of an increased level of serum calcium in the sample. An increased level of serum calcium indicates the subject is at increased risk of fatal prostate cancer.

RELATED APPLICATIONS

The present application claims the benefit of U.S. Provisional PatentApplication Ser. No. 61/141,068, filed Dec. 29, 2008, the disclosure ofwhich is incorporated by reference herein in its entirety.

GOVERNMENT SUPPORT

This invention was made with US Government Support under grant number CA014520 from the National Institutes of Health. The US Government hascertain rights to this invention.

FIELD OF THE INVENTION

The present invention concerns methods of detecting or screening forrisk of fatal prostate cancer.

BACKGROUND OF THE INVENTION

Prostate cancer is the second most fatal cancer among men in the UnitedStates, accounting for approximately 28,000 deaths in 2008 (AmericanCancer Society. Cancer Facts and Figures 2008. Atlanta: American CancerSociety; 2008 2008). Considerable epidemiologic attention in prostatecancer has focused on the vitamin D-endocrine system and on calcium(Schwartz G G. Vitamin D and the epidemiology of prostate cancer. SeminDial 2005; 18:276-89; Ahn J, Peters U, Albanes D, et al. Serum vitamin Dconcentration and prostate cancer risk: a nested case-control study. JNatl Cancer Inst 2008; 100:796-804; Tseng M. et al., Dairy, calcium, andvitamin D intakes and prostate cancer risk in the National Health andNutrition Examination Epidemiologic Follow-up Study cohort. Am J ClinNutr 2005; 81:1147-54). Although numerous studies have investigatedprostate cancer with respect to calcium intake from the diet. thesubject of calcium in serum has received scarce attention, perhapsbecause calcium levels in serum are believed to be under stricthomeostatic control. In the only study to specifically address thisquestion, we found an approximately 3-fold increased risk of fatalprostate cancer in the National Health and Nutrition Examination Survey(MANES I) Epidemiologic Follow Study (NHEFS) for men with total serumlevels of calcium in the upper tertile at the baseline exam (Skinner HG,Schwartz GG. Serum calcium and incident and fatal prostate cancer in theNational Health and Nutrition Examination Survey. Cancer EpidemiolBiomarkers Prey 2008; 17:2302-5).

SUMMARY OF THE INVENTION

A first aspect of the invention is, accordingly, a method of screeningfor increased risk of fatal prostate cancer in a human male subject,comprising: providing a blood sample collected from said subject; andthen detecting (and preferably quantitatively detecting) the presence orabsence of an increased level of serum calcium in said sample (e.g., ascompared to subjects not at increased risk of fatal prostate cancer;e.g., detecting a serum calcium level in the top tertile, or the top ormiddle tertile, of serum calcium levels for a representative populationof human male subjects)), an increased level of serum calcium indicatingsaid subject is at increased risk of fatal prostate cancer.

In some embodiments, the serum calcium detected is total serum calcium;in other embodiments, the serum calcium detected is ionized serumcalcium.

For example, when the serum calcium detected is total serum calcium, thedetecting step preferably comprises detecting a total serum calciumlevel greater than 2.3 or 2.4 mmol/L, to thereby provide an indicationof increased risk of fatal prostate cancer in the subject.

In another example, when the serum calcium is ionized serum calcium, thedetecting step preferably comprises detecting an ionized serum calciumlevel greater than 1.2 (or more particularly 1.22) or 1.3 (or moreparticularly 1.26) mmol/L to provide an indication of increased risk offatal prostate cancer in the subject.

A further aspect of the present invention is the use of a means fordetecting blood or serum calcium levels for carrying out a method ofscreening for increased risk of fatal prostate cancer in a human malesubject, such as a method described herein below.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Subjects on which the invention may be carried out are, in general,human male subjects, are typically adult subjects (e.g., greater than 18or 21 years of age), in some embodiments are at least 40 years old, andin some embodiments are geriatric subjects (e.g., greater than 65 or 70years old).

Blood samples can be collected from subjects by any suitable means,including but not limited to finger stick, venipuncture/phlebotomy, etc.Typically the blood sample is whole blood as it is withdrawn orcollected from the subject, which is then at least partially purified(e.g., to produce blood plasma or blood sample) to produce the bloodsample on which the detecting step is carried out.

The detecting step or procedure can be carried out in accordance withknown techniques, or variations thereof that will be apparent to thoseskilled in the art. In general, the detecting step is a quantitativedetecting step. Suitable detecting procedures include, but are notlimited to, absorption spectrometry (e.g., infrared absorptionspectrometry, atomic absorption spectrometry), detection with an ion(and specifically calcium) selective electrode, colorimetric detection,fluorescent detection, enzymatic detection, etc. See generally J. Henry,Clinical Diagnosis and Management by Laboratory Methods, Chap. 10, pgs.195-196 (20^(th) Ed. 2001).

Ion-selective electrodes. Ionized serum calcium can be detected with anion-selective electrode in accordance with known techniques, orvariations thereof that will be apparent to those skilled in the art.See, e.g., L. Sava et al., Indian Journal of Clinical Biochemistry20(2), 158-161 (2005); G. Bowers et al., Clin. Chem. 32/8, 1437-1447(1986). In general, such methods involve contacting the sample to anion-selective electrode; and then measuring (e.g., potentiometrically)ionized calcium in the sample through the electrode.

Atomic absorption spectrophotometry. This method can, in general, becarried out by atomizing (e.g., by a flame or heat) the sample in anatomic absorption spectrophotometer to provide an atomized sample; thenilluminating the atomized sample with light (e.g., laser light) in thespectrophotometer; then detecting light from said atomized sample with adetector; and then determining the amount of total serum calcium in saidsample from said detected light by atomic absorption spectrophotometry.Samples may be diluted with lanthanum HCl to reduce viscosity andinterference, and Strontium may be added or included as an internalstandard to correct for fluctuations in the flame and atomization rate.J. Henry, Supra at 196.

Colorimetric analysis. Total serum calcium can be colorimetricallyanalyzed by, for example, combining the sample with a metallochromicindicator dye that binds calcium to form a complex (e.g., arsenazo dyeor orthocresolphthalein complexone); and then colorimetrically measuringthe formation of the complex (e.g., with a colorimeter orspectrophotometer) to determine the amount of calcium in said sample. J.Henry, Supra. at 195.

Fluorescent analysis. Total serum calcium can be fluorescently analyzedby, for example, combining said sample with a chelating agent (e.g.,EDTA, EGTA) that binds calcium and forms a fluorescent complextherewith; and then detecting fluorescence from said sample to determinethe amount of calcium in said sample. J. Henry, Supra. at 195.

Enzymatic assay. Total serum calcium can be enzymatically determined by,for example, combining the sample with an enzyme that is eitheractivated or inhibited by calcium, and then detecting the activation orinhibition of said enzyme to determine the amount of calcium in saidsample. See, e.g., M. Sugano, Clinical Chemistry 51, 1021-1024 (2005).

Suitable enzymes include, but are not limited to, alpha amylase,phospholipase D, or urea amidolyase.

Utility. We show here that a man with a total serum calcium in the uppertertile of the normal distribution is approximately 3 times more likelyto develop fatal prostate cancer and that a man with an ionized serumcalcium in the upper tertile of the normal distribution is >3 times morelikely to develop fatal prostate cancer We envision that the greatestuse of tests such as the ionized calcium test will be to givequantitative information to men about their probability of having afatal form of prostate cancer, given a histological diagnosis ofprostate cancer. Men need this information because it would help toinform their decisions about whether to get definitive therapy (e.g.,surgery or radiation therapy) for their cancer. This is a life-alteringdecision.

Based on the two most recent years of available data from the UnitedStates' SEER (Surveillance Epidemiology and End Results) Program, thecase-fatality rate from prostate cancer (the proportion of men who diefrom prostate cancer following a diagnosis of prostate cancer) is 17.5%or roughly 1 in 6. In other words, of 6 men who today receive adiagnosis of prostate cancer, if none were treated, only 1 of the 6would die of his prostate cancer. (The others would die from causesunrelated to their prostate cancer, e.g., heart disease.) Conversely, ifall 6 were treated, 5 of the 6 would be treated unnecessarily and atconsiderable cost to their quality of life as treatment causes impotencein virtually all men, urinary and bowel incontinence (at leasttemporary) of a few percent, and death in about 1% of men who electsurgery.

Using the results described herein, we can compute the case-fatalityrates from prostate cancer within tertiles of total or ionized serumcalcium by weighting the prognosis in each tertile according to therelative risks we observed.1/3(x)+1/3(1.8) (x)+1/3(3.1)x=0.175. This yields the followingcase-fatality rates by tertile of ionized calcium:0.089, 0.160, 0.276These correspond to the following lifetime probabilities of death ineach tertile of ionized calcium: 1/11 for the lowest tertile, 1/6 forthe middle tertile, and 1/4 for the highest tertile.

Thus, the information that an ionized calcium test result could provideto men (who without the test result all start with a risk of fatalprostate cancer of 1 in 6) is that their risk is actually 50% greaterthan 1 in 6 (i.e., 1 in 4) or more than 50% lower than 1 in 6. 1 in 11.Because the decision to have definitive therapy is irrevocable onceacted upon and is life-altering, we believe that many men would wantpersonalized, numerical guidance about their probability of havingcancer that is likely to be fatal and therefore cancer that requirestreatment. For example, many men might be inclined to defer therapy ifthey learned that their risk of fatal disease was approximately 1 in 11.

Before a diagnosis of prostate cancer has been made. An importantfeature of our discovery is that the prognostic value of the testprecedes the diagnosis of prostate cancer. Because of this, we are ableto stratify men into risk categories years before a prostate canceroccurs. Men in the highest risk group may opt to have more intensivescreening, or to begin screening at an earlier age (as is done for blackmen, or men with a family history of early prostate cancer). Men in thelowest risk group may opt to delay or to forego screening, potentiallysaving themselves from the consequences of over-diagnosis and/orunnecessary treatment (for example, impotence and incontinence).

Comparison with breast cancer. In determining the utility of ourtechnology for predicting fatal prostate cancer, it may be helpful tocompare it to an analogous technology. Mutations in the BRCA1 gene areassociated with a roughly three-fold increased risk for breast cancer inwomen. This association is similar in magnitude to the association weobserve between ionized serum calcium and fatal prostate cancer. Notethat this risk is for a diagnosis of breast cancer NOT for prediction ofthe fatality of the disease (unlike our technology).

Although BRCA1 mutation is relatively rare, many women pay severalhundred to several thousand dollars to learn their BRCA1 status. In thegroup with the highest prevalence of BRCA1 mutations, women ofAshekenazi ancestry, the prevalence of BRCA1 mutations ranges from 7% inwomen with no family history of early breast or ovarian cancer to 28% inwomen with a family history of early breast and ovarian cancer. This canbe compared to 33.3% of men who fall in to the highest tertile of serumcalcium.

The importance of the information provided by this level of risk (athree-fold increase) can be measured by the choices women make when theytest positive for the mutation. Some opt for increased surveillance bymammography, some opt for chemopreventive strategies. and some opt forprophylactic surgery to remove the breasts and/or ovaries.

The present invention is explained in greater detail in the followingnon-limiting Examples.

EXAMPlE 1

METHODS. We conducted a prospective cohort study of total serum calciumand risk for incident and fatal prostate cancer using the first NationalHealth and Nutrition Examination Survey (NHANES) and the NHANESEpidemiologic Follow-up Study (National Center for Health Statistics.Plan and operation of the NHANES I Epidemiologic Follow-up Study, 1992.Washington, D.C.: DHHS, 1997; National Center for Health Statistics.Plan and operation of the NHANES I Epidemiologic Follow-up Study, 1987.Washington, D.C.: DHHS, 1992; National Center for Health Statistics.Plan and operation of the HANES I Augmentation Survey of Adults 25-74years, United States, 1974-75. Washington, D.C.: DHHS, 1978; NationalCenter for Health Statistics. Plan and operation of the Health andNutrition Examination Survey, United States, 1971-73. Washington, D.C.:DHEW, 1973). We included males aged 24 to 77 years at baselineexamination in 1971-1975. Participants who reported lung, colon, orprostate cancers prior to the baseline exam (n=102) and those whoreported prostate cancer or died within one year of baseline (n=90) wereexcluded. The final analytical cohort included 2,814 men for whombaseline serum calcium measurements were available.

Person-time at risk was calculated as the interval between the dates ofinitial examination and prostate cancer diagnosis or death (for cases)or date of last contact (for non-cases). Prostate cancer was ascertainedby self-report (with or without a recorded hospital stay) and by deathcertificate. Cases with a death record were considered fatal cases, andall others were considered non-fatal cases. For cases with a hospitalrecord, the date of diagnosis was the date of first admission forprostate cancer. For cases without hospital record the diagnosis datewas defined as June 30 of the year of self-report. For cases confirmedby death certificate only, the date of diagnosis was the date of death.The earliest diagnosis date was used for cases with multiple dates.Fatal cases were identified through death record linkage with ‘prostatecancer’ mentioned as a cause of death.

RESULTS. Eighty-five incident cases of prostate cancer, including 25fatal cases, were observed among 2,814 men over 46,188 person-years offollow-up (1971-1993). The 25 fatal cases included 10 cases for whichthe death certificate was the only record of prostate cancer. Men withhigher serum calcium tended to be younger (46 versus 52 years in the3^(rd) and 1^(st) tertiles respectively), and were more likely to beblack (15.4% versus 11.6% black in the 3^(rd) and 1^(st) tertiles).There was no evidence of increased risk of incident disease withincreasing serum calcium levels. Conversely, comparing men in the top tomen in the bottom tertile of serum calcium, we observed a significantlyincreased hazard for fatal prostate cancer that persisted afteradjustment for age (age-adjusted relative hazard of 2.59 (95% ConfidenceInterval, 1.00-6.72; P_(trend)=0.05), BMI (age-and BMI-adjusted relativehazard=2.62, Confidence Interval 1.01-6.80; P_(trend)=0.04) and race(age- and BMI- and race-adjusted relative hazard=2.68 (95% ConfidenceInterval 1.02-6.99; P_(trend)=0.04). Tests for linear trend werestatistically significant. However, most of the excess risk wasconcentrated in the top tertile of serum calcium. There was nosubstantive difference between adjusting for BMI as a continuous orcategorical variable. Adjustment for family history of prostate cancerdid not alter the point estimate but widened the confidence interval(P_(trend)=0.06). We did not detect statistically significantinteractions between serum calcium and other major risk factors forprostate cancer, but power was limited.

TABLE 1 Selected baseline characteristics of men in the NationalEpidemiologic Follow-up Study to the first National Health and NutritionExamination Survey by tertile of serum calcium. Serum Calcium Tertile 1Tertile 2 Tertile 3 Mean calcium (mg/dl) ± SD 9.3 ± 0.3 9.7 ± 0.1 10.2 ±0.3 Number of participants 992 807 1012 Age (years) 52.2 48.3 45.5 Bodymass index (kg/m²) 25.9 25.8 25.7 Race (% Black) 11.6 9.7 15.4 Familyhistory of prostate cancer (%) 0.9 1.3 1.3

TABLE 2 Relative hazards* for incident and fatal prostate cancer bytertile of serum calcium in the National Health and NutritionExamination Survey's Epidemiologic Follow-up Study. Tertile of serumcalcium (Median value) Tertile 1 Tertile 2 Tertile 3 P- (9.3 mg/dl) (9.7mg/dl) (10.1 mg/dl) trend Prostate cancer incidence Number of incidentcases 34 22 29 Person-years at risk 15556 13712 16920 Age-adjustedRelative Hazards 1.00 (Reference) 0.93 (0.54-1.60) 1.25 (0.76-2.07)0.409 +BMI 1.00 (Reference) 0.93 (0.54-1.60) 1.25 (0.76-2.07) 0.404+Race 1.00 (Reference) 0.93 (0.54-1.59) 1.29 (0.78-2.13) 0.361 +Familyhistory 1.00 (Reference) 0.97 (0.56-1.70) 1.31 (0.77-2.20) 0.341Prostate cancer mortality Number of fatal cases 7 7 11 Person-years atrisk 15556 13712 16920 Age-adjusted Relative Hazards 1.00 (Reference)1.64 (0.57-4.71) 2.59 (1.00-6.72) 0.049 +BMI 1.00 (Reference) 1.66(0.58-4.74) 2.62 (1.01-6.80) 0.046 +Race 1.00 (Reference) 1.65(0.58-4.72) 2.68 (1.02-6.99) 0.043 +Family history 1.00 (Reference) 1.72(0.55-5.37) 2.68 (0.94-7.64) 0.063 *Each model is adjusted for thelisted variable and all prior variables.

In summary, in this prospective cohort, we observed an approximately3-fold increased risk for fatal prostate cancer among men in the uppertertile of the distribution of total serum calcium. We observed asignificant dose-response. To our knowledge, this is the first study toexamine prostate cancer risk in relation to serum calcium. Themeasurement of serum calcium preceded the diagnosis of prostate cancerby an average of 9.9 years (SD=4.5 years).

EXAMPlE 2

In this example we confirm and extend the finding of Example 1 using anindependent cohort, NHANES III. A unique feature of NHANES III is thatit included measurements of ionized serum calcium, the physiologicallyactive fraction of total serum calcium.

METHODS. Data were derived the Third National Health and NutritionExamination Survey (NHANES III), which provides public-use mortalityfiles with record linkage of participants in NHANES III to the NationalDeath Index (National Center for Health Statistics (U.S.). Plan andoperation of the third National Health and Nutrition Examination Survey,1988-94. Hyattsville, Md. Washington, D.C.: U.S. Dept. of Health andHuman Services, Public Health

Service, Centers for Disease Control and Prevention; 1994). Eightthousand one hundred twenty-five (8,125) men participated in NHANES III.We excluded men with missing information on serum calcium (n=1,210); aprior history of cancer (except non-melanoma skin cancer) (n=199); menwho died from prostate cancer within 12 months of examination (n=2), andmen without follow-up information (n=4). The final analytical cohortconsisted of 6,710 men aged 18-90 years at the baseline examination in1988-1994. Because of the concern that calcium levels in serum could beinfluenced by the presence of undetected prostate cancer, we performedadditional analyses excluding prostate cancer deaths that occurredduring the first three years of follow up (N=8 deaths).

We evaluated associations between prostate cancer mortality, total serumcalcium and ionized serum calcium corrected for serum pH. Approximatelyhalf of the total serum calcium is in the ionized or physiologicallyactive state. Another 40% is bound to serum proteins, principallyalbumin, and the remaining 10% is bound to anions such as lactate andphosphate. Because the binding of calcium to proteins is altered bychanges in blood pH, measures of ionized calcium in blood commonly arepH-corrected to a standard pH using regression analysis (Dickerson R N,et al., Accuracy of methods to estimate ionized and “corrected” serumcalcium concentrations in critically ill multiple trauma patientsreceiving specialized nutrition support. JPEN J Parenter Enteral Nutr2004; 28:133-41).

Person-time was computed as the number of months from initialexamination to date of prostate cancer mortality (events), death fromanother cause, or the end of follow-up in Dec. 31, 2000 (non-events). Weestimated relative risks adjusted for potential confounders using Coxproportional hazards. Multivariate models included age (in one-yearincrements), body mass index (BMI; as a continuous measure), aspotential confounders, and variables related to NHANES III sampleweights (race/ethnicity (black/white and Hispanic/non-), household size(number of individuals up to 10), and general health status (Excellent,Very Good, Good, Fair, Poor) to account for differential probabilitiesof selection and non-response (Korn E L, Graubard B I. Epidemiologicstudies utilizing surveys: accounting for the sampling design. Am JPublic Health 1991; 81:1166-73). Statistical analysis employed SAS (v9.1for Linux) and Sudaan (v9 for Linux) to account for the complex samplingdesigns of the cohort. All statistical tests were two-sided.

RESULTS. Twenty five prostate cancer deaths occurred among 6,710 menover 56,625 person-years of follow-up. Measurement of serum calciumpreceded fatal prostate cancer by an average of 5.3 years (SD=2.5). Theprostate cancer cases' average age at baseline was 73.4 years and theiraverage age at death was 78.1 years. Men in the higher tertiles of totalserum calcium tended to be younger, to be black, to have lower BMI, tobe from a large household, and to be in good or better health. Similarrelationships were observed for increasing tertiles of ionized calcium.The mean ages in tertiles one through three were 45.2. 41.4, and 38.5years respectively.

Compared to men in the lowest tertile of total serum calcium, men in thehighest tertile had a multivariate-adjusted relative risk for prostatecancer death of 2.07 (95% Confidence

Interval, C.I.: 1.06-4.04). This association was not materially changedafter adjustment for BMI (RR=2.02; 95% C.I. 1.02-4.01). The relativerisk for men in the second tertile was 1.39 (95% C.I. 0.59-3.30). Thetest for linear trend was not statistically significant(P_(trend)=0.26). Compared to men in the lowest tertile of ionized serumcalcium, the relative risk for fatal prostate cancer among men in thehighest tertile was 3.18 (95% C.I. 1.09-9.28). Adjustment for BMI didnot substantially alter this risk (RR=3.12; 95% C.I. 1.04-9.43). Therelative risk for men in the second tertile was 1.82 (95% C.I.0.64-5.12). The linear trend was not statistically significant(P_(trend)=0.15).

TABLE 3 Selected baseline characteristics of men in the Third NationalHealth and Nutrition Examination Survey (NHANES III). 1988-1994.* TotalSerum Calcium Tertile 1 Tertile 2 Tertile 3 Number of participants 2,2052,131 2,374 Weighted population 22,801,773 23,404,681 27,861,959 Meantotal calcium (mmol/L) 2.20 2.32 2.42 Mean ionized calcium (mmol/L) 1.201.24 1.27 Age (years) 47.7 43.1 38.3 Body mass index (kg/m²) 26.7 26.926.1 Race (% Black) 8.0 10.3 11.4 Large household (% with 5 or morepeople) 36.6 41.9 45.6 Health (% Good or better) 91.2 93.4 95.0 *Meansand proportions in this table account for the complex sample design anddifferential probabilites of non-response in NHANES III and arerepresentative of the total population of U.S. men. NOTE: To converttotal calcium or ionized calcium from mmol/L to mg/dL, divide mmol/L by0.2495

In summary, we found a doubling of risk for fatal prostate cancer amongmen in the highest tertile of total serum calcium and a tripling of riskfor men in the highest tertile of ionized serum calcium. The results fortotal serum calcium are consistent with our findings for prostate cancermortality in NHANES I in which we observed a multi-variable adjusted RRof 2.68. This is the first study to examine prostate cancer risk inrelation to prediagnostic levels of ionized serum calcium.

TABLE 4 Relative hazards* for prostate cancer mortality by tertiles oftotal and ionized serum calcium in the Third National Health andNutrition Examination Survey (NHANES III) Tertile of serum calcium P-Tertile 1 Tertile 2 Tertile 3 trend Total Serum Calcium (mmol/L) Median(Range) 2.22 2.31 2.41 mmol/L (1.06-2.27) (2.28-2.35) (2.36-3.06) Numberof deaths 9 8 8 Person-months at risk 218,964 216,797 243,725Age-adjusted Relative 1.00 1.39 2.07 0.258 Hazards (Reference)(0.59-3.30) (1.06-4.04) + Body Mass Index 1.00 1.36 2.02 0.287(Reference) (0.57-3.26) (1.02-4.01) Ionized Serum Calcium (mmol/L)Median (Range) 1.19 1.24 1.28 mmol/L (0.99-1.21) (1.22-1.25) (1.26-1.60)Number of deaths 6 8 11 Person-months at risk 199,120 252,180 228,186Age-adjusted Relative 1.00 1.82 3.18 0.152 Hazards (Reference)(0.64-5.12) (1.09-9.28) + Body Mass Index 1.00 1.81 3.12 0.160(Reference) (0.63-5.20) (1.04-9.43) *In addition to adjustement for age(one-year intervals) and body mass index (as a continuous measure), allmodels are adusted for variables used in NHANES III sample weighting: aninteraction between senior status (age > 60 years) and race/ethnicity,household size, and general health status(Excellent/VeryGood/Good/Fair/Poor). Models account for clustering ofobservations in complex sampling design of NHANES III. Ionized serumcalcium is pH corrected.

The foregoing is illustrative of the present invention, and is not to beconstrued as limiting thereof. The invention is defined by the followingclaims, with equivalents of the claims to be included therein.

That which is claimed is:
 1. A method of screening for increased risk ofdeveloping fatal prostate cancer in a human male subject in needthereof, comprising: providing a blood sample collected from saidsubject; and detecting the presence or absence of an increased level ofserum calcium in said sample, an increased level of serum calciumindicating said subject is at increased risk of fatal prostate cancer;wherein said serum calcium is total serum calcium and said increasedlevel is greater than 2.3 mmol/L, and said detecting step is carried outby absorption spectrometry.
 2. The method of claim 1, wherein said bloodsample comprises whole blood, blood plasma, or blood serum.
 3. Themethod of claim 1, wherein said providing step comprises collecting ablood sample from said subject.
 4. The method of claim 1, wherein saidproviding step comprises at least partially purifying said blood sample.5. The method of claim 1, wherein said detecting step is a quantitativedetecting step.
 6. A method of screening for increased risk ofdeveloping fatal prostate cancer in a human male subject in needthereof, comprising: providing a blood sample collected from saidsubject; and detecting the presence or absence of an increased level ofserum calcium in said sample, an increased level of serum calciumindicating said subject is at increased risk of fatal prostate cancer;wherein said serum calcium is total serum calcium and said increasedlevel is greater than 2.3 mmol/L, and said detecting step is carried outby: atomizing said sample in an atomic absorption spectrophotometer toprovide an atomized sample; then illuminating said atomized sample withlight in said atomic absorption spectrophotometer; then detecting lightfrom said atomized sample with a detector; and then determining theamount of total serum calcium in said sample from said detected light byatomic absorption spectrophotometry.
 7. The method of claim 6, whereinsaid blood sample comprises whole blood, blood plasma, or blood serum.8. The method of claim 6, wherein said providing step comprisescollecting a blood sample from said subject.
 9. The method of claim 6,wherein said providing step comprises at least partially purifying saidblood sample.
 10. The method of claim 6, wherein said detecting step isa quantitative detecting step.